Monoclonal antibodies are traditionally made by establishing an immortal mammalian cell line which is derived from a single immunoglobulin producing cell secreting one form of a biologically functional antibody molecule with a particular specificity. Because the antibody-secreting mammalian cell line is immortal, the characteristics of the antibody are reproducible from batch to batch. The key proprieties of monoclonal antibodies are their specificity for a particular antigen and the reproducibility with which they can be manufactured.
Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds. The light chains exist in two distinct forms called K (kappa) and .lambda. (lambda). Each chain has a constant region (C) and a variable region (V). Each chain is organized into a series of domains. The light chains have two domains, corresponding to the C region and the other to the V Region. The heavy chains have four domains, one corresponding to the V region and three domains (1, 2, and 3) in the C region. The antibody has two arms (each arm being a Fab region), each of which has a VL and a VH region associated with each other. It is this pair of V regions (VL and VH) that differ from one antibody to another (owing to amino acid sequence variations), and which together are responsible for recognizing the antigen and providing an antigen binding site (ABS). In even more detail, each V region is made up from three complementarily determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of the variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via a complex process involving recombination, mutation, and selection.
It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) the dAb fragment (Ward et al., Nature 341 (1989): 544-546) which consists of a VH domain; (v) isolated CDR regions; and (vi) F(ab').sub.2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
Although the two domains of the Fv fragment are coded for by separate genes, it has proved possible to make a synthetic linker that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al., Science 242 (1988):423-426; Huston et al., Proc. Natl. Acad. Sci., USA 85 (1988):5879-5883); by recombinant methods. These scFv fragments were assembled from genes from monoclonals that had been previously isolated. In our earlier application, WO 92/01047, we described a process to assemble scFv fragments from VH and VL domains that were not part of a previously isolated antibody.
Although monoclonal antibodies, their fragments and derivatives have been enormously advantageous, there are nevertheless a number of limitations associated with them. First, the therapeutic applications of monoclonal antibodies produced by human immortal cell lines holds great promise for the treatment of a wide range of diseases (Lennox, Clinical Applications of Monoclonal Antibodies, British Medical Bulletin 1984). Unfortunately, immortal antibody-producing human cell lines are very difficult to establish and they give low yields of antibody (approximately 1 .mu.g/ml). In contrast, equivalent rodent cell lines yield high amounts of antibody (approximately 100 .mu.g/ml). However, the repeated administration of these foreign rodent proteins to humans can lead to harmful hypersensitivity reactions. As a result, these rodent-derived monoclonal antibodies have limited therapeutic use.
Second, a key aspect in the isolation of monoclonal antibodies is how many different clones of antibody producing cells with different specificities, can be practically established and sampled compared to how many theoretically need to be sampled in order to isolate a cell producing antibody with the desired specificity characteristics (Milstein, Royal Soc. Croonian Lecture, Proc. R. Soc. London B. 239 (1990):1-16). For example, the number of different specificities expressed at any one time by lymphocytes of the murine immune system is thought to be approximately 10.sup.7 and this is only a small proportion of the potential repertoire of specificities. However, during the isolation of a typical antibody producing cell with a desired specificity, the investigator is only able to sample 10.sup.3 to 10.sup.4 individual specificities. The problem is worse in the human, where one has approximately 10.sup.12 lymphocyte specificities with the limitation on sampling of 10.sup.3 or 10.sup.4 remaining.
This problem has been alleviated to some extent in laboratory animals by the use of immunization regimes. Thus, where one wants to produce monoclonal antibodies having a specificity against a particular epitope, an animal is immunized with an immunogen expressing that epitope. The animal will then mount an immune response against the immunogen and there will be a proliferation of lymphocytes which have specificity against the epitope. Owing to this proliferation of lymphocytes with the desired specificity, it becomes easier to detect them in the sampling procedure. However, this approach is not successful in all cases as a suitable immunogen may not be available. Furthermore, where one wants to produce human monoclonal antibodies (e.g., for therapeutic administration), such an approach is not practically or ethically feasible.
In our earlier application, WO 92/01047, we described methods of constructing a bacteriophage that expresses and displays at its surface a large biologically functional binding molecule (e.g., antibody fragments, enzymes, and receptors) and which remains intact and infectious. We called the structure which comprises a virus particle and a binding molecule displayed at the viral surface a "package". Where the binding molecule is an antibody, an antibody derivative or fragment, or a domain that is homologous to an immunoglobulin domain, we called the package a "phage antibody" (pAb). However, except where the context demanded otherwise, where the term phage antibody is used generally, it was also interpreted as referring to any package comprising a virus particle and a biologically functional binding molecule displayed at the viral surface. Since the original filing of WO 92/01047, a number of examples of functional antibody and other protein domains expressed on the surface of bacteriophage have been reported in both the literature and additional patent applications.
This simple substitution of immortalized cells with bacterial cells as the "factory", considerably simplifies procedures for preparing large amounts of binding molecules expressed on the surface of the bacteriophage. Furthermore, the use of polymerase chain reaction (PCR) amplification (Saiki et al., Science 239 (1988):487-491) to isolate antibody producing sequences from cells (e.g., hybridomas and B cells) has great potential for speeding up the timescale under which binding specificities can be isolated. Phage antibody expression libraries can be easily generated by cloning the amplified VH and VL genes directly into bacteriophage expression vectors. Furthermore, a bacteriophage based recombinant production system allows scope for producing tailor-made antibodies and fragments thereof. For example, it is possible to produce chimeric molecules with new combinations of binding and effector functions, humanized antibodies (e.g., murine variable regions combined with human constant domains or murine-antibody CDRs grafted onto a human FR) and novel antigen-binding molecules. The key advantage of the phage based system being the ability to directly screen the recombinant antibodies directly for the desired binding specificities.
In creating recombinant VH and VL phage libraries several problems need to be addressed. For example, in a mouse there are approximately 10.sup.7 possible H chains and 10.sup.7 possible L chains. Therefore, there are 10.sup.14 possible combinations of H and L chains, and to test for anything like this number of combinations, one would have to create and screen a library of about 10.sup.14 clones. This had not previously been a practical possibility. PCT/GB92/00883 and PCT/GB92/01755 applications, which are herein incorporated by reference, disclose a number of approaches which ameliorate this problem. Each of these applications is a continuation-in-part of our International Application WO 92/01047.
In addition, a number of molecular biological techniques which have previously been developed for engineering of antibody active sites can be applied in combination with the phage antibody library approaches described previously. These techniques include site-directed mutagenesis of residues within a CDR, replacement of all or portions of CDR (s) with random amino acid sequence, CDR shuffling in which a CDR region is essentially replaced with a library of CDR regions. The use of pAbs may also allow the construction of entirely synthetic antibodies. Furthermore, antibodies may be made which have some synthetic sequences, for example, CDRs, and some naturally derived sequences (see for example PCT/BG92/06372). For example, V-gene repertoires could be made in vitro by combining un-rearranged V genes, with D and J segments. Libraries of pAbs could then be selected by binding to antigen, hypermutated in vitro in the antigen-binding loops or V domain framework regions, and subjected to further rounds of selection and mutagenesis.
pAbs have a range of applications in selecting antibody genes encoding antigen binding activities. One particularly exciting area of application is in the development of antibodies with catalytic properties (catalytic antibodies). Catalytic antibodies have been described in U.S. Pat. Nos. 4,888,281 to Schochetman et al.; 4,963,355 to Kim et al.; and 5,037,750 to Schochetman et al., all hereby incorporated by reference. As disclosed therein, catalytic antibodies combine the catalytic abilities of enzymes with the binding capabilities of antibodies.
All catalytic antibodies described to date have been generated using monoclonal antibody technology. The details of that process are well known to those of ordinary skill in the art. A typical methodology first involves immunizing mice with an appropriate antigen. The antigen may be the desired reactant; the desired reactant bound to a peptide or other carrier molecule; a reaction intermediate or an analog of the reactant; or the product or a reaction intermediate.
"Analog" as the term is used here can encompass isomers, homologs, transition state analogs or other compounds sufficiently resembling the reactant in terms of chemical structure such that an antibody raised against the analog may participate in an immunological reaction with the reactant but will not necessarily catalyze a reaction of the analog.
Although a number of different types of antibody catalysts have been developed with this technology, the time required to establish and then screen the hybridomas for the desired specificity is of considerable importance.
If the desired specificity is sufficiently rare, it may be impractical or impossible to sample enough hybridomas cell lines to recover the desired specificity.
Additionally, there is currently no suitable hybridoma based technology for generating entirely human catalytic antibodies.
The methods of the invention can also be used to effect a cleavage that leads to the activation of some biological function.
Such reactions include the cleavage of peptide bonds, but may also include ester bonds or glycosidic bonds or other types of bonds.
One example of the cleavage of a biomolecule which leads to the activation of a biological function is the treatment of insulin-dependent diabetes. Patients self-administer insulin by injection. Prior attempts to develop a formulation of insulin whose release into the circulation mimics the pharmacokinetics of the release of natural pancreatic insulin have not proved successful. Insulin exists in the pancreas in a pro-form, proinsulin, whose activity is many orders of magnitude lower than insulin itself. An antibody protease specific for the peptide bond that leads to conversion of proinsulin to insulin can be designed so that its kinetic characteristics allow release of insulin in vivo after an injection of proinsulin plus antibody protease. This is an example of prodrug activation where the drug in this instance is a natural protein hormone. Prodrugs may include many therapeutically active molecules which lead to the activation or deactivation of a biological function. The pro-form may either take advantage of a natural modification of the drug or any suitable synthetic modification thereof. Suitable drug derivatives with low activity (therapeutically beneficial or toxic), which, on modification with a suitable catalytic antibody, are converted to an active form. A particular example of this process is given in PCT/US89/01951 filed May 4, 1989, which is hereby incorporated by reference.